Screening DNA Damage in the Rat Kidney and Liver by Untargeted DNA Adductomics.

Air pollution, tobacco smoke, and red meat are associated with renal cell cancer (RCC) risk in the United States and Western Europe; however, the chemicals that form DNA adducts and initiate RCC are mainly unknown. Aristolochia herbaceous plants are used for medicinal purposes in Asia and worldwide. They are a significant risk factor for upper tract urothelial carcinoma (UTUC) and RCC to a lesser extent. The aristolochic acid (AA) 8-methoxy-6-nitrophenanthro-[3,4-d]-1,3-dioxolo-5-carboxylic acid (AA-I), a component of Aristolochia herbs, contributes to UTUC in Asian cohorts and in Croatia, where AA-I exposure occurs from ingesting contaminated wheat flour. The DNA adduct of AA-I, 7-(2'-deoxyadenosin-N6-yl)-aristolactam I, is often detected in patients with UTUC, and its characteristic A:T-to-T:A mutational signature occurs in oncogenes and tumor suppressor genes in AA-associated UTUC. Identifying DNA adducts in the renal parenchyma and pelvis caused by other chemicals is crucial to gaining insights into unknown RCC and UTUC etiologies. We employed untargeted screening with wide-selected ion monitoring tandem mass spectrometry (wide-SIM/MS2) with nanoflow liquid chromatography/Orbitrap mass spectrometry to detect DNA adducts formed in rat kidneys and liver from a mixture of 13 environmental, tobacco, and dietary carcinogens that may contribute to RCC. Twenty DNA adducts were detected. DNA adducts of 3-nitrobenzanthrone (3-NBA), an atmospheric pollutant, and AA-I were the most abundant. The nitrophenanthrene moieties of 3-NBA and AA-I undergo reduction to their N-hydroxy intermediates to form 2'-deoxyguanosine (dG) and 2'-deoxyadenosine (dA) adducts. We also discovered a 2'-deoxycytidine AA-I adduct and dA and dG adducts of 10-methoxy-6-nitro-phenanthro-[3,4-d]-1,3-dioxolo-5-carboxylic acid (AA-III), an AA-I isomer and minor component of the herbal extract assayed, signifying AA-III is a potent kidney DNA-damaging agent. The roles of AA-III, other nitrophenanthrenes, and nitroarenes in renal DNA damage and human RCC warrant further study. Wide-SIM/MS2 is a powerful scanning technology in DNA adduct discovery and cancer etiology characterization.

Loss of proximal tubular transcription factor Krüppel-like factor 15 exacerbates kidney injury through loss of fatty acid oxidation.

Loss of fatty acid ß-oxidation (FAO) in the proximal tubule is a critical mediator of acute kidney injury and eventual fibrosis. However, transcriptional mediators of FAO in proximal tubule injury remain understudied. Krüppel-like factor 15 (KLF15), a highly enriched zinc-finger transcription factor in the proximal tubule, was significantly reduced in proximal tubule cells after aristolochic acid I (AAI) treatment, a proximal tubule-specific injury model. Proximal tubule specific knockout of Klf15 exacerbated proximal tubule injury and kidney function decline compared to control mice during the active phase of AAI treatment, and after ischemia-reperfusion injury. Furthermore, along with worsening proximal tubule injury and kidney function decline, knockout mice exhibited increased kidney fibrosis as compared to control mice during the remodeling phase after AAI treatment. RNA-sequencing of kidney cortex demonstrated increased transcripts involved in immune system and integrin signaling pathways and decreased transcripts encompassing metabolic pathways, specifically FAO, and PPARα signaling, in knockout versus control mice after AAI treatment. In silico and experimental chromatin immunoprecipitation studies collectively demonstrated that KLF15 occupied the promoter region of key FAO genes, CPT1A and ACAA2, in close proximity to transcription factor PPARα binding sites. While the loss of Klf15 reduced the expression of Cpt1a and Acaa2 and led to compromised FAO, induction of KLF15 partially rescued loss of FAO in AAI-treated cells. Klf15, Ppara, Cpt1a, and Acaa2 expression was also decreased in other mouse kidney injury models. Tubulointerstitial KLF15 independently correlated with eGFR, PPARA and CPT1A appearance in expression arrays from human kidney biopsies. Thus, proximal tubule-specific loss of Klf15 exacerbates acute kidney injury and fibrosis, likely due to loss of interaction with PPARα leading to loss of FAO gene transcription.

Aristolochic acid and its effect on different cancers in uro-oncology.

PURPOSE OF REVIEW: To acquaint urologists with aristolochic acid nephropathy, an iatrogenic disease that poses a distinct threat to global public health. In China alone, 100 million people may currently be at risk. We illustrate the power of molecular epidemiology in establishing the cause of this disease. RECENT FINDINGS: Molecular epidemiologic approaches and novel mechanistic information established a causative linkage between exposure to aristolochic acid and urothelial carcinomas of the bladder and upper urinary tract. Noninvasive tests are available that detect urothelial cancers through the genetic analysis of urinary DNA. Combined with cytology, some of these tests can detect 95% of patients at risk of developing bladder and/or upper urothelial tract cancer. Robust biomarkers, including DNA-adduct and mutational signature analysis, unequivocally identify aristolochic acid-induced tumours. The high mutational load associated with aristolochic acid-induced tumours renders them candidates for immune-checkpoint therapy. SUMMARY: Guided by recent developments that facilitate early detection of urothelial cancers, the morbidity and mortality associated with aristolochic acid-induced bladder and upper tract urothelial carcinomas may be substantially reduced. The molecular epidemiology tools that define aristolochic acid-induced tumours may be applicable to other studies assessing potential environmental carcinogens.

Genome-wide quantification of rare somatic mutations in normal human tissues using massively parallel sequencing.

We present the bottleneck sequencing system (BotSeqS), a next-generation sequencing method that simultaneously quantifies rare somatic point mutations across the mitochondrial and nuclear genomes. BotSeqS combines molecular barcoding with a simple dilution step immediately before library amplification. We use BotSeqS to show age- and tissue-dependent accumulations of rare mutations and demonstrate that somatic mutational burden in normal human tissues can vary by several orders of magnitude, depending on biologic and environmental factors. We further show major differences between the mutational patterns of the mitochondrial and nuclear genomes in normal tissues. Lastly, the mutation spectra of normal tissues were different from each other, but similar to those of the cancers that arose in them. This technology can provide insights into the number and nature of genetic alterations in normal tissues and can be used to address a variety of fundamental questions about the genomes of diseased tissues.

Method for Biomonitoring DNA Adducts in Exfoliated Urinary Cells by Mass Spectrometry.

Tobacco smoking contributes to about 50% of the bladder-cancer (BC) cases in the United States. Some aromatic amines in tobacco smoke are bladder carcinogens; however, other causal agents of BC are uncertain. Exfoliated urinary cells (EUCs) are a promising noninvasive biospecimen to screen for DNA adducts of chemicals that damage the bladder genome, although the analysis of DNA adducts in EUCs is technically challenging because of the low number of EUCs and limiting quantity of cellular DNA. Moreover, EUCs and their DNA adducts must remain viable during the time of collection and storage of urine to develop robust screening methods. We employed RT4 cells, a well-differentiated transitional epithelial bladder cell line, as a cell-model system in urine to investigate cell viability and the chemical stability of DNA adducts of two prototypical bladder carcinogens: 4-aminobiphenyl (4-ABP), an aromatic amine found in tobacco smoke, and aristolochic acid I (AA-I), a nitrophenanthrene found in Aristolochia herbaceous plants used for medicinal purposes worldwide. The cell viability of RT4 cells pretreated with 4-ABP or AA-I in urine exceeded 80%, and the major DNA adducts of 4-ABP and AA-I, quantified by liquid chromatography-mass spectrometry, were stable for 24 h. Thereafter, we successfully screened EUCs of mice treated with AA-I to measure DNA adducts of AA-I, which were still detected 25 days following treatment with the carcinogen. EUCs are promising biospecimens that can be employed for the screening of DNA adducts of environmental and dietary genotoxicants that may contribute to the development of BC.

Targeted and Untargeted Detection of DNA Adducts of Aromatic Amine Carcinogens in Human Bladder by Ultra-Performance Liquid Chromatography-High-Resolution Mass Spectrometry.

Epidemiological studies have linked aromatic amines (AAs) from tobacco smoke and some occupational exposures with bladder cancer risk. Several epidemiological studies have also reported a plausible role for structurally related heterocyclic aromatic amines present in tobacco smoke or formed in cooked meats with bladder cancer risk. DNA adduct formation is an initial biochemical event in bladder carcinogenesis. We examined paired fresh-frozen (FR) and formalin-fixed paraffin-embedded (FFPE) nontumor bladder tissues from 41 bladder cancer patients for DNA adducts of 4-aminobiphenyl (4-ABP), a bladder carcinogen present in tobacco smoke, and 2-amino-9 H-pyrido[2,3- b]indole, 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine and 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline, possible human carcinogens, which occur in tobacco smoke and cooked meats. These chemicals are present in urine of tobacco smokers or omnivores. Targeted DNA adduct measurements were done by ultra-performance liquid chromatography-electrospray ionization multistage hybrid Orbitrap MS. N-(2'-Deoxyguanosin-8-yl)-4-ABP ( N-(dG-C8)-4-ABP) was the sole adduct detected in FR and FFPE bladder tissues. Twelve subjects (29%) had N-(dG-C8)-4-ABP levels above the limit of quantification, ranging from 1.4 to 33.8 adducts per 109 nucleotides (nt). DNA adducts of other human AA bladder carcinogens, including 2-naphthylamine (2-NA), 2-methylaniline (2-MA), 2,6-dimethylaniline (2,6-DMA), and lipid peroxidation (LPO) adducts, were screened for in bladder tissue, by our untargeted data-independent adductomics method, termed wide-selected ion monitoring (wide-SIM)/MS2. Wide-SIM/MS2 successfully detected N-(dG-C8)-4-ABP, N-(2'-deoxyadenosin-8-yl)-4-ABP and the presumed hydrazo linked adduct, N-(2'-deoxyguanosin- N2-yl)-4-ABP, and several LPO adducts in bladder DNA. Wide-SIM/MS2 detected multiple DNA adducts of 2-NA, 2-MA, and, 2,6-DMA, when calf thymus DNA was modified with reactive intermediates of these carcinogens. However, these AA-adducts were below the limit of detection in unspiked human bladder DNA (<1 adduct per 108 nt). Wide-SIM/MS2 can screen for many types of DNA adducts formed with exogenous and endogenous electrophiles and will be employed to identify DNA adducts of other chemicals that may contribute to the etiology of bladder cancer.

A Rapid Throughput Method To Extract DNA from Formalin-Fixed Paraffin-Embedded Tissues for Biomonitoring Carcinogenic DNA Adducts.

Formalin-fixed paraffin-embedded (FFPE) tissues are rarely used for screening DNA adducts of carcinogens because the harsh conditions required to reverse the formaldehyde-mediated DNA cross-links can destroy DNA adducts. We recently adapted a commercial silica-based column kit used in genomics to manually isolate DNA under mild conditions from FFPE tissues of rodents and humans and successfully measured DNA adducts of several carcinogens including aristolochic acid I (AA-I), 4-aminobiphenyl (4-ABP), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (Yun et al. (2013) Anal. Chem. 85, 4251-8, and Guo et al. (2016) Anal. Chem. 88, 4780-7). The DNA retrieval methodology is robust; however, the procedure is time-consuming and labor intensive, and not amenable to rapid throughput processing. In this study, we have employed the Promega Maxwell 16 MDx system, which is commonly used in large scale genomics studies, for the rapid throughput extraction of DNA. This system streamlines the DNA isolation procedure and increases the sample processing rate by about 8-fold over the manual method (32 samples versus 4 samples processed per hour). High purity DNA is obtained in satisfactory yield for the measurements of DNA adducts by ultra performance liquid chromatography-electrospray-ionization-ion trap-multistage scan mass spectrometry. The measurements show that the levels of DNA adducts of AA-I, 4-ABP, and PhIP in FFPE rodent and human tissues are comparable to those levels measured in DNA from matching tissues isolated by the commercial silica-based column kits and in DNA from fresh frozen tissues isolated by the conventional phenol-chloroform extraction method. The isolation of DNA from tissues is one major bottleneck in the analysis of DNA adducts. This rapid throughput methodology greatly decreases the time required to process DNA and can be employed in large-scale epidemiology studies designed to assess the role of chemical exposures and DNA adducts in cancer risk.

Multiclass Carcinogenic DNA Adduct Quantification in Formalin-Fixed Paraffin-Embedded Tissues by Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry.

DNA adducts are a measure of internal exposure to genotoxicants and an important biomarker for human risk assessment. However, the employment of DNA adducts as biomarkers in human studies is often restricted because fresh-frozen tissues are not available. In contrast, formalin-fixed paraffin-embedded (FFPE) tissues with clinical diagnosis are readily accessible. Recently, our laboratory reported that DNA adducts of aristolochic acid, a carcinogenic component of Aristolochia herbs used in traditional Chinese medicines worldwide, can be recovered quantitatively from FFPE tissues. In this study, we have evaluated the efficacy of our method for retrieval of DNA adducts from archived tissue by measuring DNA adducts derived from four other classes of human carcinogens: polycyclic aromatic hydrocarbons (PAHs), aromatic amines, heterocyclic aromatic amines (HAAs), and N-nitroso compounds (NOCs). Deoxyguanosine (dG) adducts of the PAH benzo[a]pyrene (B[a]P), 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (dG-N(2)-B[a]PDE); the aromatic amine 4-aminobiphenyl (4-ABP), N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-4-ABP); the HAA 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), N-(deoxyguanosin-8-yl)-PhIP (dG-C8-PhIP); and the dG adducts of the NOC 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), O(6)-methyl-dG (O(6)-Me-dG) and O(6)-pyridyloxobutyl-dG (O(6)-POB-dG), formed in liver, lung, bladder, pancreas, or colon were recovered in comparable yields from fresh-frozen and FFPE preserved tissues of rodents treated with the procarcinogens. Quantification was achieved by ultraperformance liquid chromatography coupled with electrospray ionization ion-trap multistage mass spectrometry (UPLC/ESI-IT-MS(3)). These advancements in the technology of DNA adduct retrieval from FFPE tissue clear the way for use of archived pathology samples in molecular epidemiology studies designed to assess the causal role of exposure to hazardous chemicals with cancer risk.

TERT promoter mutations occur frequently in gliomas and a subset of tumors derived from cells with low rates of self-renewal.

Malignant cells, like all actively growing cells, must maintain their telomeres, but genetic mechanisms responsible for telomere maintenance in tumors have only recently been discovered. In particular, mutations of the telomere binding proteins alpha thalassemia/mental retardation syndrome X-linked (ATRX) or death-domain associated protein (DAXX) have been shown to underlie a telomere maintenance mechanism not involving telomerase (alternative lengthening of telomeres), and point mutations in the promoter of the telomerase reverse transcriptase (TERT) gene increase telomerase expression and have been shown to occur in melanomas and a small number of other tumors. To further define the tumor types in which this latter mechanism plays a role, we surveyed 1,230 tumors of 60 different types. We found that tumors could be divided into types with low (<15%) and high (≥15%) frequencies of TERT promoter mutations. The nine TERT-high tumor types almost always originated in tissues with relatively low rates of self renewal, including melanomas, liposarcomas, hepatocellular carcinomas, urothelial carcinomas, squamous cell carcinomas of the tongue, medulloblastomas, and subtypes of gliomas (including 83% of primary glioblastoma, the most common brain tumor type). TERT and ATRX mutations were mutually exclusive, suggesting that these two genetic mechanisms confer equivalent selective growth advantages. In addition to their implications for understanding the relationship between telomeres and tumorigenesis, TERT mutations provide a biomarker that may be useful for the early detection of urinary tract and liver tumors and aid in the classification and prognostication of brain tumors.

Aristolochic acid-induced apoptosis and G2 cell cycle arrest depends on ROS generation and MAP kinases activation.

Ingestion of aristolochic acids (AAs) contained in herbal remedies results in a renal disease and, frequently, urothelial malignancy. The genotoxicity of AA in renal cells, including mutagenic DNA adducts formation, is well documented. However, the mechanisms of AA-induced tubular atrophy and renal fibrosis are largely unknown. To better elucidate some aspects of this process, we studied cell cycle distribution and cell survival of renal epithelial cells treated with AAI at low and high doses. A low dose of AA induces cell cycle arrest in G2/M phase via activation of DNA damage checkpoint pathway ATM-Chk2-p53-p21. DNA damage signaling pathway is activated more likely via increased production of reactive oxygen species (ROS) caused by AA treatment then via DNA damage induced directly by AA. Higher AA concentration induced cell death partly via apoptosis. Since mitogen-activated protein kinases play an important role in cell survival, death and cell cycle progression, we assayed their function in AA-treated renal tubular epithelial cells. ERK1/2 and p38 but not JNK were activated in cells treated with AA. In addition, pharmacological inhibition of ERK1/2 and p38 as well as suppression of ROS generation with N-acetyl-L-cysteine resulted in the partial relief of cells from G2/M checkpoint and a decline of apoptosis level. Cell cycle arrest may be a mechanism for DNA repair, cell survival and reprogramming of epithelial cells to the fibroblast type. An apoptosis of renal epithelial cells at higher AA dose might be necessary to provide space for newly reprogrammed fibrotic cells.

Formalin-fixed paraffin-embedded tissue as a source for quantitation of carcinogen DNA adducts: aristolochic acid as a prototype carcinogen.

DNA adducts are a measure of internal exposure to genotoxicants. However, the measurement of DNA adducts in molecular epidemiology studies often is precluded by the lack of fresh tissue. In contrast, formalin-fixed paraffin-embedded (FFPE) tissues frequently are accessible, although technical challenges remain in retrieval of high quality DNA suitable for biomonitoring of adducts. Aristolochic acids (AA) are human carcinogens found in Aristolochia plants, some of which have been used in the preparation of traditional Chinese herbal medicines. We previously established a method to measure DNA adducts of AA in FFPE tissue. In this study, we examine additional features of formalin fixation that could impact the quantity and quality of DNA and report on the recovery of AA-DNA adducts in mice exposed to AA. The yield of DNA isolated from tissues fixed with formalin decreased over 1 week; however, the levels of AA-DNA adducts were similar to those in fresh frozen tissue. Moreover, DNA from FFPE tissue served as a template for PCR amplification, yielding sequence data of comparable quality to DNA obtained from fresh frozen tissue. The estimates of AA-DNA adducts measured in freshly frozen tissue and matching FFPE tissue blocks of human kidney stored for 9 years showed good concordance. Thus, DNA isolated from FFPE tissues may be used to biomonitor DNA adducts and to amplify genes used for mutational analysis, providing clues regarding the origin of human cancers for which an environmental cause is suspected.

Folate, homocysteine and the cardiac neural crest.

Congenital heart defects (CHD) are the most common congenital defects worldwide, and perigestational folate supplementation (PFS) is the most effective large-scale intervention to date for reducing CHD. This review is based upon the following premises: that the majority of CHD result from disruption of development of the cardiac neural crest (CNC); and that the CNC is highly responsive to folate and homocysteine. The following roles of folate are discussed in relation to CNC development: one-carbon metabolism in support of mitosis and gene methylation; and gene regulation via direct activity of the folate receptor. The following roles of hyperhomocysteinemia are discussed in the same context: increased oxidative stress; disruption of gene methylation; homocysteinylation of key proteins; and NMDA receptor binding. It is proposed that well-focused advances in folate-CNC research could lead to development of strategies, in addition to PFS, to facilitate normal CNC and heart development, and thereby further reduce CHD.

Mechanisms of antiviral action and toxicities of ipecac alkaloids: Emetine and dehydroemetine exhibit anti-coronaviral activities at non-cardiotoxic concentrations.

The emergence of highly infectious pathogens with their potential for triggering global pandemics necessitate the development of effective treatment strategies, including broad-spectrum antiviral therapies to safeguard human health. This study investigates the antiviral activity of emetine, dehydroemetine (DHE), and congeneric compounds against SARS-CoV-2 and HCoV-OC43, and evaluates their impact on the host cell. Concurrently, we assess the potential cardiotoxicity of these ipecac alkaloids. Significantly, our data reveal that emetine and the (-)-R,S isomer of 2,3-dehydroemetine (designated in this paper as DHE4) reduce viral growth at nanomolar concentrations (i.e., IC50 ∼ 50-100 nM), paralleling those required for inhibition of protein synthesis, while calcium channel blocking activity occurs at elevated concentrations (i.e., IC50 ∼ 40-60 µM). Our findings suggest that the antiviral mechanisms primarily involve disruption of host cell protein synthesis and is demonstrably stereoisomer specific. The prospect of a therapeutic window in which emetine or DHE4 inhibit viral propagation without cardiotoxicity renders these alkaloids viable candidates in strategies worthy of clinical investigation.

Human formalin-fixed paraffin-embedded tissues: an untapped specimen for biomonitoring of carcinogen DNA adducts by mass spectrometry.

DNA adducts represent internal dosimeters to measure exposure to environmental and endogenous genotoxicants. Unfortunately, in molecular epidemiologic studies, measurements of DNA adducts often are precluded by the unavailability of fresh tissue. In contrast, formalin-fixed paraffin embedded (FFPE) tissues frequently are accessible for biomarker discovery. We report here that DNA adducts of aristolochic acids (AAs) can be measured in FFPE tissues at a level of sensitivity comparable to freshly frozen tissue. AAs are nephrotoxic and carcinogenic compounds found in Aristolochia herbaceous plants, many of which have been used worldwide for medicinal purposes. AAs are implicated in the etiology of aristolochic acid nephropathy and upper urinary tract carcinoma. 8-Methoxy-6-nitrophenanthro-[3,4-d]-1,3-dioxole-5-carboxylic acid (AA-I) is a component of Aristolochia herbs and a potent human urothelial carcinogen. AA-I reacts with DNA to form the aristolactam (AL-I)-DNA adduct 7-(deoxyadenosin-N(6)-yl) aristolactam I (dA-AL-I). We established a method to quantitatively retrieve dA-AL-I from FFPE tissue. Adducts were measured, using ultraperformance liquid chromatography/mass spectrometry, in liver and kidney tissues of mice exposed to AA-I, at doses ranging from 0.001 to 1 mg/kg body weight. dA-AL-I was then measured in 10-µm thick tissue-sections of FFPE kidney from patients with upper urinary tract cancers; the values were comparable to those observed in fresh frozen samples. The limit of quantification of dA-AL-I was 3 adducts per 10(9) DNA bases per 2.5 µg of DNA. The ability to retrospectively analyze FFPE tissues for DNA adducts may provide clues to the origin of human cancers for which an environmental cause is suspected.

A fluorescence-based analysis of aristolochic acid-derived DNA adducts.

Aristolochic acids (AAs), major components of plant extracts from Aristolochia species, form (after metabolic activation) pro-mutagenic DNA adducts in renal tissue. The DNA adducts can be used as biomarkers for studies of AA toxicity. Identification of these adducts is a complicated and time-consuming procedure. We present here a fast, nonisotopic, fluorescence-based assay for the detection of AA-DNA adducts in multiple samples. This approach allows analysis of AA adducts in synthetic DNA with known nucleotide composition and analysis of DNA adducts formed from chemically diverse AAs in vitro. The method can be applied to compare AA-DNA adduct formation in cells and tissues.

Biomonitoring of aristolactam-DNA adducts in human tissues using ultra-performance liquid chromatography/ion-trap mass spectrometry.

Aristolochic acids (AAs) are a structurally related family of nephrotoxic and carcinogenic nitrophenanthrene compounds found in Aristolochia herbaceous plants, many of which have been used worldwide for medicinal purposes. AAs have been implicated in the etiology of so-called Chinese herbs nephropathy and of Balkan endemic nephropathy. Both of these disease syndromes are associated with carcinomas of the upper urinary tract (UUC). 8-Methoxy-6-nitrophenanthro-[3,4-d]-1,3-dioxolo-5-carboxylic acid (AA-I) is a principal component of Aristolochia herbs. Following metabolic activation, AA-I reacts with DNA to form aristolactam (AL-I)-DNA adducts. We have developed a sensitive analytical method, using ultraperformance liquid chromatography-electrospray ionization/multistage mass spectrometry (UPLC-ESI/MS(n)) with a linear quadrupole ion-trap mass spectrometer, to measure 7-(deoxyadenosin-N(6)-yl) aristolactam I (dA-AL-I) and 7-(deoxyguanosin-N(2)-yl) aristolactam I (dG-AL-I) adducts. Using 10 µg of DNA for measurements, the lower limits of quantitation of dA-AL-I and dG-AL-I are, respectively, 0.3 and 1.0 adducts per 10(8) DNA bases. We have used UPLC-ESI/MS(n) to quantify AL-DNA adducts in tissues of rodents exposed to AA and in the renal cortex of patients with UUC who reside in Taiwan, where the incidence of this uncommon cancer is the highest reported for any country in the world. In human tissues, dA-AL-I was detected at levels ranging from 9 to 338 adducts per 10(8) DNA bases, whereas dG-AL-I was not found. We conclude that UPLC-ESI/MS(n) is a highly sensitive, specific and robust analytical method, positioned to supplant (32)P-postlabeling techniques currently used for biomonitoring of DNA adducts in human tissues. Importantly, UPLC-ESI/MS(n) could be used to document exposure to AA, the toxicant responsible for AA nephropathy and its associated UUC.

MESD is essential for apical localization of megalin/LRP2 in the visceral endoderm.

Deletion of the Mesd gene region blocks gastrulation and mesoderm differentiation in mice. MESD is a chaperone for the Wnt co-receptors: low-density lipoprotein receptor-related protein (LRP) 5 and 6 (LRP5/6). We hypothesized that loss of Wnt signaling is responsible for the polarity defects observed in Mesd-deficient embryos. However, because the Mesd-deficient embryo is considerably smaller than Lrp5/6 or Wnt3 mutants, we predicted that MESD function extends more broadly to the LRP family of receptors. Consistent with this prediction, we demonstrated that MESD function in vitro was essential for maturation of the ß-propeller/EGF domain common to LRPs. To begin to understand the role of MESD in LRP maturation in vivo, we generated a targeted Mesd knockout and verified that loss of Mesd blocks WNT signaling in vivo. Mesd mutants continue to express the pluripotency markers Oct4, Nanog, and Sox2, suggesting that Wnt signaling is essential for differentiation of the epiblast. Moreover, we demonstrated that MESD was essential for the apical localization of the related LRP2 (Megalin/MEG) in the visceral endoderm, resulting in impaired endocytic function. Combined, our results provide evidence that MESD functions as a general LRP chaperone and suggest that the Mesd phenotype results from both signaling and endocytic defects resulting from misfolding of multiple LRP receptors.

Genetic loci that affect aristolochic acid-induced nephrotoxicity in the mouse.

Aristolochic acids (AA) are plant-derived nephrotoxins and carcinogens found in traditional medicines and herbal remedies. AA causes aristolochic acid nephropathy (AAN) and is a suspected environmental agent in Balkan endemic nephropathy (BEN) and its associated upper urothelial cancer. Approximately 5-10% of individuals exposed to AA develop renal insufficiency and/or cancer; thus a genetic predisposition to AA sensitivity has been proposed. The mouse is an established animal model of AAN, and inbred murine strains vary in AA sensitivity, confirming the genetic predisposition. We mapped quantitative trait loci (QTL) correlated with proximal tubule dysfunction after exposure to AA in an F2 population of mice, derived from breeding an AA-resistant strain (C57BL/6J) and an AA-sensitive strain (DBA/2J). A single main QTL was identified on chromosome 4 (Aanq1); three other interacting QTLs, (Aanq2-4) also were detected. The Aanq1 region was also detected in untreated mice, raising the possibility that preexisting differences in proximal tubule function may affect the severity of AA-elicited toxicity. This study lays the groundwork for identifying the genetic pathways contributing to AA sensitivity in the mouse and will further our understanding of human susceptibility to AA found widely in traditional medicines.

High-affinity folate receptor in cardiac neural crest migration: a gene knockdown model using siRNA.

Folate supplementation reduces the incidence of congenital heart defects, but the nature of this protective mechanism remains unclear. Immunolabeling demonstrated that the neural tube and neural crest (NC) cells were rich in the high-affinity folate receptor FOLR1and during the early stages of development FOLR1 was found principally in these cells. Suppression of Folr1 expression in the nascent cardiac NC by site-directed short-interfering RNA (siRNA) altered cardiac NC cell mitosis and subsequent migration patterns leading to abnormal development of the pharyngeal arch arteries (PAA) and outflow tract. qPCR analysis demonstrated that the siRNA treatment significantly reduced Folr1 24 hr after treatment. These treatments also significantly reduced mitosis in the neural tube, but adjacent, nontreated areas were unaffected. In summary, a brief reduction in the expression of Folr1 during a critical stage of NC development had long-term consequences for the development of the PAA and outflow tract.